This invention relates to methods and apparatus for reducing Schlieren noise in a liquid chromatograph.
It is known to reduce Schlieren noise in a liquid chromatograph by reducing the temperature differences between the eluate and the flow cell. This reduces the difference in density and hence differences in refraction index between portions of the eluate in the flow cell. Since the eluate is in motion, such differences deflect light in an unpredictable manner, thus producing photometric noise.
The prior art technique attempts to maintain a temperature equality between the flow cell and the eluate entering the flow cell by introducing the eluate into a heat exchanger built into the flow cell. In this technique, the outlet of the heat exchanger is connected to the flow entrance of the flow cell and the body of the heat exchanger is thermally connected to the body of the flow cell. Another technique is to use a conical optical path in the flow cell so that Schlieren scattered light is not absorbed by the cell walls.
The prior art techniques have several disadvantages, such as: (1) a substantial amount of Schlieren noise remains; (2) the absorbance detector causes heat to be conducted to the flow cell and therefore sets up temperature gradients within the flow cell with respect to the flow of eluate; (3) the techniques are slow to stabilize and require much time before measurements can be made without excessive Schlieren noise; and (4) the fluid volume of the heat exchanger or the excess volume of the conical optical path over a cylindrical path degrades the degree of separation of narrow chromatographic peaks or bands in the eluate.
The disadvantage caused by the fluid volume of the heat exchanger or the excess volume of the conical optical path over a cylindrical path is a particular problem with micro-liquid chromatography. A common column diameter for micro-liquid chromatography (MLC) is one millimeter and a common conventional high performance liquid chromatography (HPLC) column diameter is 4.6 millimeters. Thus, the cross sectional area of a MLC column is about one-twentieth (1/20) that of an HPLC column, and so MLC solute peaks are smaller in volume by at least a factor of 20. It is more than a factor of 20 if the length of a peak residing in the column also decreases when going from HPLC to MLC.
Because the solute peaks are smaller in MLC, the peak resolution degradation problems are aggravated because of the difficulty of detecting and collecting low volume bands. This problem is further aggravated when micro-capillary MLC columns with a diameter of 0.1 millimeter or less are used.
The smaller peak volumes of MLC require proportionately smaller fluid volumes in the optical path in order to maintain the same chromatographic resolution. Since shortening the optical path length decreases the absorbance sensitivity according to Beer's Law, the diameter of the path length must be made smaller instead. The greater length to diameter ratio of MLC cells increases their Schlieren noise over that of comparable HPLC cells, thus making it even more of a problem with MLC than with HPLC. The conventional HPLC noise reduction methods are undesirable for MLC because they degrade volumetric resolution which is much more critical for MLC than for HPLC.